Amylase production by a Schwanniomyces occidentalis mutant in chemostat culture

The crystalline cell surface layer (S-layer) from Bacillus stearothermophilis PV72 was used as a matrix for reversible immobilization of -d-galactosidase via disulphide bonds. In order to obtain an immobilization matrix stable towards acid, alkali and reducing agents such as dithiothreitol (DTT), the S-layer subunits were first cross-linked with glutaraldehyde. This was done in a way whereby 75% of the free amino groups remained unmodified, and then could be completely converted into sulphhydryl groups upon reaction with the monofunctional imidoester iminothiolane. After activation of the sulphhydryl groups with 2,2-dipyridyldisulphide, 550 g -d-galactosidase could be immobilized per milligram of S-layer protein, which corresponds to one -d-galactosidase molecule [relative molecular mass (M_r), 116000] per two S-layer subunits (M_r, 130 000). At least 90% of the sulphhydryl groups from the S-layer protein could be regenerated for further activation by cleaving the disulphide bonds with DTT. In comparative studies -d-galactosidase was linked to carbodiimide-activated carboxyl groups of the S-layer protein.Correspondence to: M. Sára